Kinetic constants determination for an alkaline protease from Bacillus mojavensis using response surface methodology

Abstract

The kinetic constants for an alkaline protease from Bacillus mojavensis were determined using a central composite circumscribed design (CCCD) where concentration of substrate (casein) and the assay temperature were varied around their center point. The K_m,V_max, K_cat, activation energy (E_a) and temperature coefficient (q₁₀) were determined and the values of these kinetic constants obtained were found comparable to that obtained with conventional methods. The Michaelis-Menten constant (K_m) for casein decreased with corresponding increase in V_max, as reaction temperature was raised from 45–60°C. The protease exhibited K_m of 0.0357 mg/ml, 0.0270 mg/ml, 0.0259 mg/ml, and 0.0250 mg/ml at 45, 50, 55, and 60°C, respectively, whereas V_max values at these temperatures were 74.07, 99.01, 116.28, and 120.48 μg/ml/min, respectively, as determined by response surface methodology. The Arrhenius plot suggested that the enzyme undergoes thermal activation above 45°C until 60–65°C followed by thermal inactivation. Likewise, the energy of activation (E_a) was more between 45–55°C (9747 cal/mol) compared to E_a between 50–60°C (4162 cal/mol). © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 289–295, 2002.